The pattern of activity in the circuits of the brain and their experience-dependent changes underlie the processing of sensory information, perception, and motor control. Much has been learned about the anatomical wiring of brain circuits and about the properties of individual neurons in the intact brain, but considerable mystery remains about how the properties of individual neurons emerge from their connectivity and how multiple groups of neurons are activated during behaviors. Part of the problem has been that high precision electrical recording is usually obtained from only one or a few neurons at a time, when salient events are actually processed by large assemblies of neurons. To provide for a fast high-resolution recording from mammalian neurons, this proposal seeks to improve fluorescent protein (FP) based voltage sensors. These probes will be self-contained, not requiring any exogenous factors to function, and thus will be genetically-encodable. We are seeking probes that are readily expressed on the cell's surface, show maximum changes in intensity with membrane potential alterations, respond rapidly to changes in membrane potential, and are minimally disruptive to cells. Members of the project have been involved in the development of first generation FP-voltage sensors, including Fluorescent Shaker (FlaSh), Voltage-Sensitive Fluorescent Protein (VSFP) and Sodium channel Protein Activity Reporting Construct (SPARC). These constructs have demonstrated the feasibility of creating channel-FP constructs that alter fluorescence intensity with changes in cell membrane potential. Significant improvements in the response characteristics may come from a pseudo-saturating examination of the ion channel/transporter and fluorescent protein space. This proposal will create large libraries of membrane protein / FP fusion constructs varying the membrane protein, the location of the inserted FP and the isoform of the inserted FP. These libraries will be created by a novel transposon-based FP insertion process. Constructs will be screened for surface expression in hippocampal neurons and tested for voltage-dependent fluorescence changes using fast fluorescence measurements combined with voltage-clamp electrophysiology. We will express the most promising FP-voltage sensors in brain slices and in vivo using viral infection followed by the production of transgenic mice.